We propose to study the regulation of expression of chromosomally encoded multidrug resistance (MDR) efflux pumps and their roles in antimicrobial resistance in Staphylococcus aureus, with a focus on quinolone resistance. In prior work we identified quinolone resistance caused for increased expression of the NorA, B, and C pumps, tetracycline resistance due to the Tet38 pump, (-lactam resistance due to the AbcA pump, and others have identified biocide resistance due to the MepA pump. We have also identified direct transcriptional regulators of pump expression, MgrA and NorG, which interact with each other and have broad effects. There are five Specific Aims. Under Aim 1, we will define the interactions of MgrA and NorG in expression of norA and other genes encoding efflux pumps. We specifically hypothesize that phosphorylation of MgrA alters its interactions with NorG and changes its effects on transcription of pump genes. The work will include measuring expression of norA by Northern hybridization in strains with constructed mutations in HprK kinase, and measurements of promoter binding of phosphorylated and unphosphorylated MgrA. Under Aim 2, we will identify the direct regulators of norB, tet38, and abcA by isolating proteins from cell extracts that bind to immobilized promoter DNA fragments, determining their identity by mass spectroscopy, and generating strains with mutations in and overexpression of the genes so identified. Such strains will be assessed for changes in expression of genes encoding pumps by Northern hybridization and transcriptional profiling. Under Aim 3, we will evaluate the global effects of NorG by transcriptional profiling of strains with mutations in and overexpression of norG. Under Aim 4, we will identify other candidate efflux pumps shown to be over-expressed in microarray analyses of regulatory networks and environmental stresses and define their roles in antibiotic resistance. Specific novel candidate pump genes distinct from those listed have been shown to be overexpressed in sarA, mgrA, and rot global regulatory mutants and under conditions evoking the stringent response or growth in a mouse subcutaneous abscess model. These candidate pump genes will be cloned and the effects of their overexpression on susceptibility to antibiotics and biocides will be assessed in order to add to the complement of known efflux resistance mechanisms. Under Aim 5, we will identify the environmental triggers of increased expression of norB, tet38, and other efflux pump genes identified under Aim 4. We have shown that norB and tet38 are overexpressed in S. aureus abscesses and that knockouts of these genes reduce fitness in the abscess milieu. Thus, we hypothesize that pumps are important for bacterial survival in an abscess in part due to relative protection from antimicrobial peptides, which are present in high concentrations. We will test specifically the role of low iron conditions and high concentrations of antimicrobial peptides as inducers of pump expression by measurement of pump gene RNA levels by Northern blotting, and we will determine the effects of pump overexpression of resistance to antimicrobial peptides in vitro. PUBLIC HEALTH RELEVANCE Antibiotic resistance in common bacterial pathogens such as Staphylococcus aureus is increasing in hospitals and the community. Efflux pumps serve to protect bacteria from adverse environments. Multidrug (MDR) and quinolone resistance conferred by multidrug resistance efflux pumps can also compromise drug utility and affect patient responses to antibiotics. Thus, defining the full complement of MDR efflux pumps in S. aureus and how they are regulated will provide both practical and fundamental information that are important for understanding of staphylococcal fitness to cause disease in diverse environments and for optimizing antibiotic action and use.